The primary goal of this project is to better characterize the interaction of hepatitis C virus (HCV) with the host cell in an effort to overcome the bottleneck to HCV research that is posed by the absence of permissive ell culture systems. In Specific Aim 1, we will utilize modified dicistronic and monocistronic versions of an infectious molecular HCV clone that encode a positive selectable marker to assess viral determinants of replication in cultured cells. We will determine whether these modified RNAs remain infectious in chimpanzees, and can be selected for replication in cultured cells under increasing antibiotic pressure. Long range goals include the recloning of HCV cDNA from cell cultures supporting replication of these RNAs, in order to identify mutations associated with adaptation to replication in a new cellular environment. Substantial preliminary data support the feasibility of these experiments. In Specific Aim 2, we will assess the replication competence of candidate subgenomic dicistronic replicons expressing selectable markers in transfected cells, and use such replicons to explore the cellular determinants of permissiveness for HCV replication. We will determine the extend to which the selection of clonal cell lines supporting replicon amplification reflects adaptation of the viral RNA to the cell culture system, and whether co-transfection with a human liver expression library enables to promote replication in stably transformed cell lines. We have shown that polypyrimidine tract-binding protein (PTB) up-regulates HCV translation in vivo, and thus will also assess its ability to enable replicon amplification. Since other preliminary data indicate that expression of one or more HCV proteins may lead to altered and assess the impact of the forced expression of Bcl-2 on survival and growth of cells transfected with HCV. In Specific Aim 3, we will utilize high density oligonucleotide microarrays to determine the impact of HCV HCV structural proteins at different levels of abundance. These experiments will be carried in collaboration with Project 2, and should add to our understanding of the restrictions to HCV replication in cultured cells.